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Forensic Toxicology
Published in: Forensic Toxicology 2/2015

01-07-2015 | Original Article

Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry

Authors: Marisol S. Castaneto, Ariane Wohlfarth, Shaokun Pang, Mingshe Zhu, Karl B. Scheidweiler, Robert Kronstrand, Marilyn A. Huestis

Published in: Forensic Toxicology | Issue 2/2015

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Abstract

AB-FUBINACA, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide, is an indazole synthetic cannabinoid identified in drug seizures around the world. Few metabolism data are available, despite the need for human urinary markers to detect AB-FUBINACA intake. Our main objective was to identify suitable analytical targets by analyzing human hepatocyte incubation samples with high-resolution mass spectrometry (HRMS) and to confirm the results in authentic urine specimens. We also determined AB-FUBINACA’s metabolic stability in human liver microsomes (HLMs) and compared hepatocyte and urine results with in silico predictions. The metabolic stability of AB-FUBINACA was determined in pooled HLMs (1 µmol/l, up to 1 h). The metabolite profile of human hepatocytes (10 µmol/l, 1 and 3 h) and urine samples from two subjects were determined by HRMS using information-dependent tandem-mass spectrometry (MS-MS) acquisition. Data were analyzed with MetabolitePilot™ software utilizing different processing algorithms, including generic peak finding, mass defect filtering, neutral loss, and product ion filtering. In silico metabolite prediction was performed with MetaSite™ software. AB-FUBINACA’s half-life in HLMs was 62.6 ± 4.0 min. AB-FUBINACA produced 11 metabolites (2 glucuronides) in human hepatocytes and 10 were identified in authentic human urine. Major metabolic pathways were terminal amide hydrolysis, acyl glucuronidation and hydroxylation at the aminooxobutane moiety. Epoxidation followed by hydrolysis, hydroxylation at the indazole moiety and dehydrogenation were minor pathways. Defluorination did not occur. Seventeen first-generation metabolites were predicted in silico, of which seven were observed in vitro and eight in vivo. We recommend AB-FUBINACA carboxylic acid, hydroxy AB-FUBINACA carboxylic acid, dihydrodiol AB-FUBINACA and dihydrodiol AB-FUBINACA carboxylic acid as suitable urinary markers.
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Metadata
Title
Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry
Authors
Marisol S. Castaneto
Ariane Wohlfarth
Shaokun Pang
Mingshe Zhu
Karl B. Scheidweiler
Robert Kronstrand
Marilyn A. Huestis
Publication date
01-07-2015
Publisher
Springer Japan
Published in
Forensic Toxicology / Issue 2/2015
Print ISSN: 1860-8965
Electronic ISSN: 1860-8973
DOI
https://doi.org/10.1007/s11419-015-0275-8

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